Method for detecting 2, 5-dichloro-4, 6-dimethyl nicotinonitrile related substances of starting material of olpicopone

Abstract

The invention relates to a method for detecting related substances of 2, 5-dichloro-4, 6-dimethyl nicotinazonitrile as a starting material of olpilcapone, which adopts high performance liquid chromatography for detection, and adopts octadecylsilane chemically bonded silica as a filler as a chromatographic column. The detector is an ultraviolet detector; the mobile phase A is a phosphoric acid solution; the mobile phase B is acetonitrile; a gradient elution mode is adopted. According to the present invention, the high performance liquid chromatography method capable of effectively separating the 2, 5-dichloro-4, 6-dimethyl nicotinonitrile and various impurities is established for the first time, 9 impurities can be simultaneously and effectively separated, the purity of the material can be accurately, conveniently and rapidly detected, the side reaction during the synthesis process of the olpicopone can be reduced from the source, the yield can be easily increased, and the production cost can be easily reduced; the detection method disclosed by the invention has good specificity, sensitivity, precision and durability, and provides a reliable means for quality control of the starting material of the olpicopone, so that the quality of an olpicopone bulk drug product is effectively guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of chemical drug analysis technology, specifically relating to a high-performance liquid chromatography method for the detection of related substances in 2,5-dichloro-4,6-dimethylnicotinonitrile, the starting material of opipicolane. Background Technology

[0002] Opicapone is a new generation of peripheral catechol O-methyltransferase (COMT) inhibitors developed by the Portuguese company Bial-Portela. Opicapone capsules are marketed as Ongentys. It was first approved in the EU in 2016 as adjunctive therapy to levodopa / dopa decarboxylase inhibitors for the treatment of Parkinson's disease patients experiencing end-of-dose symptoms that are not stably controlled. It was approved in the US in 2020 for adjunctive therapy to levodopa / carbidopa for Parkinson's disease patients experiencing the "on-off" phenomenon. It is currently available in several countries.

[0003] Opipapone, with the molecular formula C0, has the following molecular formula: 15 H 10 Cl2N4O6, with a molecular weight of 413.17, has the following structural formula:

[0004]

[0005] In the industrial synthesis of opipcarbon, 2,5-dichloro-4,6-dimethylnicotinonitrile is a commonly used key starting material with the molecular formula C8H6Cl2N2, a molecular weight of 201.05, and the structural formula as follows:

[0006]

[0007] As a crucial structural fragment of opipicolane, strictly controlling the related substances of 2,5-dichloro-4,6-dimethylnicotinonitrile is essential for reducing side reactions during opipicolane synthesis and ensuring the quality of the final drug substance. However, no methods for separating and detecting related impurities in this starting material have been reported. Therefore, there is an urgent need in the field to develop a method for effectively separating and detecting 2,5-dichloro-4,6-dimethylnicotinonitrile and its related impurities. Summary of the Invention

[0008] The purpose of this invention is to provide a method for detecting related substances in 2,5-dichloro-4,6-dimethylnicotinonitrile, the starting material of oppicarbon. This method can effectively separate the main component and various impurities, and can accurately, conveniently and quickly detect the purity of the material, thereby reducing side reactions in the oppicarbon synthesis process from the source. The detection method of this invention has good specificity, sensitivity, precision and robustness.

[0009] In one aspect, this invention provides a method for detecting related substances in the opiacarbon starting material 2,5-dichloro-4,6-dimethylnicotinonitrile, using high-performance liquid chromatography (HPLC). The HPLC conditions include:

[0010] Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel;

[0011] Detector: Ultraviolet detector;

[0012] Mobile phase: Mobile phase A is phosphoric acid solution; Mobile phase B is acetonitrile;

[0013] Elution method: gradient elution.

[0014] Preferably, the related substances have an ultraviolet absorption wavelength in the range of 190-400 nm. Specifically, the related substances include impurity I, impurity II, impurity III, impurity IV, impurity V, impurity VI, impurity VII, impurity VIII, and impurity IX.

[0015]

[0016] Preferably, the mobile phase A is a 0.05%-0.15% (V / V) phosphoric acid solution, more preferably a 0.09-0.11% (V / V) phosphoric acid solution, and most preferably a 0.1% (V / V) phosphoric acid solution;

[0017] Preferably, the gradient elution procedure is as follows:

[0018]

[0019]

[0020] More preferably,

[0021]

[0022] Preferably, the chromatographic column is a Waters Xselect HSS T3 with dimensions of 4.6 mm × 250 mm and 5 μm or 4.6 mm × 250 mm and 3.5 μm.

[0023] Preferably, the flow rate of the mobile phase is 0.9–1.1 ml / min;

[0024] Preferably, the column temperature of the chromatographic column is 25–35°C;

[0025] Preferably, the detection wavelength is 210-218 nm;

[0026] Preferably, the injection volume for the detection is 10–30 μl;

[0027] Preferably, the detection method further includes the following solution preparation step:

[0028] Preparation of diluent: It is prepared by mixing water and acetonitrile, preferably by mixing water and acetonitrile in a volume ratio of 1:1;

[0029] Preparation of test solution: Take 2,5-dichloro-4,6-dimethylnicotinonitrile and dilute quantitatively with diluent to prepare a solution containing approximately 0.2 mg per ml;

[0030] Preparation of the reference solution: Take 2,5-dichloro-4,6-dimethylnicotinonitrile reference standard and dilute it quantitatively with diluent to prepare a solution containing approximately 0.4 μg per 1 ml;

[0031] Preparation of system suitability solution: Take 2,5-dichloro-4,6-dimethylnicotinonitrile and each impurity reference standard, and dilute quantitatively with diluent to prepare a mixed solution containing approximately 0.2 mg of 2,5-dichloro-4,6-dimethylnicotinonitrile and 0.4 μg of each impurity per 1 ml;

[0032] Preferably, the detection method further includes an analytical method verification step, including the preparation of an APKP001 localization solution: take 2,5-dichloro-4,6-dimethylnicotinonitrile (APKP001) reference standard, and quantitatively dilute it with a diluent to prepare a solution containing approximately 0.2 mg per 1 ml;

[0033] Preferably, the detection method further includes an analytical method verification step, including the preparation of an impurity stock solution: taking each impurity reference standard and quantitatively diluting it with a diluent to prepare a solution containing approximately 0.2 mg per 1 ml;

[0034] Preferably, the detection method further includes an analytical method verification step, including the preparation of impurity localization solutions: taking each impurity stock solution and diluting it with a diluent to a solution containing approximately 0.4 μg per 1 ml;

[0035] Preferably, the detection method further includes an analytical method verification step, including the preparation of a detection limit test solution: take 1 ml of APKP001 positioning solution and 1 ml of each impurity stock solution, place them in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain an intermediate stock solution; dilute the intermediate stock solution stepwise with diluent until the peak height signal-to-noise ratio (S / N) of each component is approximately 3.

[0036] Preferably, the content of the related substances is calculated using the principal component self-comparison method with a correction factor, and the calculation formula is as follows:

[0037] Impurity content (%) = f × A 杂 / A 对 ×C 对 / C 供 ×100;

[0038] In the formula: f is the correction factor for each impurity;

[0039] A 杂 The peak area of ​​each impurity peak in the test solution;

[0040] A 对 The peak area of ​​the main component peak in the control solution;

[0041] C 对 The concentration of the control solution is (mg / ml).

[0042] C 供 The concentration of the test solution is (mg / ml).

[0043] The present invention also provides the application of the above-described detection method in the quality control of opipcarbon or its starting materials.

[0044] The beneficial effects of this invention are as follows:

[0045] 1. This invention establishes for the first time a high-performance liquid chromatography method that can effectively separate 2,5-dichloro-4,6-dimethylnicotinonitrile and various impurities, thus solving the quality control problem of this material.

[0046] 2. The detection method of this invention can simultaneously and effectively separate up to nine impurities. It accurately, conveniently, and rapidly detects the purity of 2,5-dichloro-4,6-dimethylnicotinonitrile, reducing side reactions in the synthesis of opipcarbon from the source, which helps to improve yield, reduce production costs, and ultimately ensure the quality control of opipcarbon raw materials.

[0047] 3. Verification has shown that the detection method of the present invention has good specificity, sensitivity (detection limit up to 0.01%), precision (RSD < 10%) and robustness, meeting the requirements of drug quality research, and providing a reliable means for the quality control of opipicolane starting materials, thereby effectively ensuring the quality of opipicolane API products. Attached Figure Description

[0048] Figure 1 The HPLC chromatogram of the diluent in Example 1 is shown below.

[0049] Figure 2 The HPLC chromatogram of the test solution in Example 1;

[0050] Figure 3 The HPLC chromatogram of the system suitability solution in Example 1 is shown.

[0051] Figure 4 This is the HPLC chromatogram of the detection limit concentration in Example 1. Detailed Implementation

[0052] The present invention will be further described in detail below through embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the structural concept and scope of use of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0053] Example 1

[0054] High performance liquid chromatograph: Waters e2695-2489;

[0055] Chromatographic column: Waters Xselect HSS T3, 4.6mm × 250mm, 5μm;

[0056] Flow rate: 1.0 ml / min, column temperature: 30℃, detection wavelength: 210 nm, injection volume: 20 μl;

[0057] Mobile phase A: 0.1% (V / V) phosphoric acid solution;

[0058] Mobile phase B: Acetonitrile;

[0059] Perform linear gradient elution according to Table 1:

[0060] Table 1 Elution gradient

[0061]

[0062] Experimental steps:

[0063] Prepare the diluent: Measure 1L of water and 1L of acetonitrile, mix, and shake well.

[0064] Preparation of APKP001 positioning solution: Weigh approximately 10 mg of 2,5-dichloro-4,6-dimethylnicotinonitrile (APKP001) reference standard accurately, place it in a 50 ml volumetric flask, add 2 ml of acetonitrile to dissolve it, dilute to the mark with diluent, and shake well;

[0065] Preparation of impurity stock solutions: Weigh approximately 10 mg each of impurity I, impurity II, impurity III, impurity IV, impurity V, impurity VI, impurity VII, impurity IX, and impurity VIII reference standards, place them in separate 50 ml volumetric flasks, dissolve them in an appropriate amount of acetonitrile, dilute to the mark with diluent, and shake well to obtain the impurity stock solutions.

[0066] Preparation of impurity localization solutions: Transfer 0.2 ml of each impurity stock solution to a 100 ml volumetric flask, dilute to the mark with diluent, and mix well.

[0067] Preparation of system suitability solution: Weigh approximately 20 mg of 2,5-dichloro-4,6-dimethylnicotinonitrile (APKP001) reference standard accurately, place it in a 100 ml volumetric flask, dissolve it with an appropriate amount of acetonitrile, and then measure 0.2 ml of each impurity stock solution into the same volumetric flask. Dilute to the mark with diluent and shake well.

[0068] Preparation of test solution: Weigh approximately 20 mg of the APGP001 test sample accurately, place it in a 100 ml volumetric flask, add 2 ml of acetonitrile to dissolve it, then dilute to the mark with diluent and shake well.

[0069] Preparation of the control solution: Weigh approximately 20 mg of the APGP001 reference standard accurately, place it in a 100 ml volumetric flask, add 2 ml of acetonitrile to dissolve it, and then dilute quantitatively with diluent to prepare a solution containing approximately 0.4 μg per ml.

[0070] Preparation of detection limit test solution: Accurately measure 1 ml each of APKP001 positioning solution and each impurity stock solution, place them in the same 100 ml volumetric flask, dilute to the mark with diluent, shake well to obtain intermediate stock solution; accurately measure an appropriate amount of intermediate stock solution, dilute stepwise with diluent until the peak signal-to-noise ratio (S / N) of each component is about 3, this sample solution is used as the detection limit test solution for each component.

[0071] Experiment 1: Specificity Experiment

[0072] Inject 20 μl each of the above-mentioned diluent, APKP001 positioning solution, impurity positioning solution, system suitability solution, test solution, control solution, and detection limit investigation solution into the liquid chromatograph, and record the chromatograms for each. Figure 1 HPLC chromatogram of the diluent; Figure 2 HPLC chromatogram of the test solution; Figure 3 HPLC chromatograms of the solution for system suitability; Figure 4 HPLC chromatogram for the detection limit concentration.

[0073] Results of specificity test:

[0074] Table 2 Results of Specificity Examination

[0075]

[0076]

[0077] according to Figure 1 , Figure 2 , Figure 3As shown in Table 2, the diluent did not interfere with the detection of the test components; the minimum resolution between the components was 1.5.

[0078] Experiment 2: Sensitivity Test

[0079] The detection limit for known impurities is determined based on the signal-to-noise ratio (SNR). This involves comparing the signal measured from a known low-concentration sample with the signal from a blank sample to calculate the lowest concentration or amount of the analyte that can be reliably detected. Generally, the detection limit is defined as the concentration at which the SNR is approximately 3.

[0080] Sensitivity test results:

[0081] Table 3 Detection Limit Results

[0082]

[0083] according to Figure 4 As shown in Table 3, the signal-to-noise ratio of impurities with a concentration of 0.01% in the test sample solution is above 3, ensuring that all related substances above 0.01% in the test sample can be detected. The impurity detection capability of this method is 0.4 ng, proving that the detection sensitivity of this method is high.

[0084] Experiment 3: Precision Test

[0085] The precision of the method is evaluated by examining the repeatability and intermediate precision of the following two sets of experiments.

[0086] (1) Repeatability

[0087] Test solution: Weigh approximately 20 mg of the APKP001 test sample accurately, place it in a 100 ml volumetric flask, add 2 ml of acetonitrile to dissolve it, then dilute to the mark with diluent and shake well.

[0088] Control solution: Weigh approximately 20 mg of the APGP001 reference standard accurately, place it in a 100 ml volumetric flask, add 2 ml of acetonitrile to dissolve it, and then dilute quantitatively with diluent to prepare a solution containing approximately 0.4 μg per ml.

[0089] Take the test solution and the control solution, and inject them for detection according to the chromatographic conditions of Example 1.

[0090] (2) Intermediate precision

[0091] Another researcher retested the six test solutions and corresponding control solutions on different dates using different chromatographic systems, following a repeatability method. The results of the precision test are shown in Tables 4 and 5 below.

[0092] Table 4 Precision Results (Experimenter A)

[0093]

[0094] Table 5 Precision Results (Experimenter B)

[0095]

[0096]

[0097] According to the results in Tables 4 and 5, the repeatability of the six test solutions from experimenters A and B shows that the RSDs of impurities IV and IX, other individual impurities, and the total amount of impurities are all less than 10.0%, and impurities I, II, III, V, VI, VII, and VIII were not detected in the test solutions. For the twelve test solutions, impurities I, II, III, V, VI, VII, and VIII were not detected, and the RSDs of impurities IV and IX, other individual impurities, and the total amount of impurities are all less than 10.0%, demonstrating the good precision of this method.

[0098] Test 4: Durability Test

[0099] The impact of changes in chromatographic conditions on the analytical method will be assessed by altering the phosphoric acid concentration of mobile phase A, column temperature, flow rate, and using different numbered columns of the same brand and specifications.

[0100] The diluent, system suitability solution, test solution and control solution were prepared according to Example 1, and the samples were injected and tested according to the robustness chromatographic conditions. The robustness results are shown in Table 6 below.

[0101] Table 6 Durability Results

[0102]

[0103]

[0104] According to the results in Table 6, under all the experimental conditions (changing the phosphoric acid concentration of mobile phase A (±0.01% (V / V)), flow rate (±0.1 ml / min), column temperature (±5℃), and using different numbered columns of the same brand and specifications), impurities I, II, III, V, VI, VII, VIII, and other individual impurities were not detected. The RSDs of impurities IV, IX, and the total amount of impurities were all less than 10.0%, demonstrating the good robustness of this method.

[0105] While the embodiments disclosed in this invention are as described above, the content is merely for the purpose of facilitating understanding of the invention and is not intended to limit the invention. Any person skilled in the art to which this invention pertains may make any modifications and variations in the form and details of the implementation without departing from the spirit and scope disclosed herein; however, the scope of patent protection of this invention shall still be determined by the scope defined in the appended claims.

Claims

1. A method for detecting related substances in the opiacarbon starting material 2,5-dichloro-4,6-dimethylnicotinonitrile, characterized in that, The detection was performed using high-performance liquid chromatography (HPLC), and the HPLC conditions included: Chromatographic column: A chromatographic column packed with octadecylsilane-bonded silica gel; Detector: Ultraviolet detector; Mobile phase: Mobile phase A is phosphoric acid solution; Mobile phase B is acetonitrile; Elution method: gradient elution.

2. The detection method as described in claim 1, characterized in that, The relevant substances include impurities I, II, III, IV, V, VI, VII, VIII, and IX:

3. The detection method as described in claim 1 or 2, characterized in that, The mobile phase A is a 0.05%-0.15% (V / V) phosphoric acid solution, preferably a 0.09-0.11% (V / V) phosphoric acid solution.

4. The detection method according to any one of claims 1-3, characterized in that, The gradient elution procedure is as follows: Preferred, 5. The detection method according to any one of claims 1-4, characterized in that, The chromatographic column is a Waters Xselect HSS T3 with dimensions of 4.6 mm × 250 mm and 5 μm or 4.6 mm × 250 mm and 3.5 μm.

6. The detection method according to any one of claims 1-5, characterized in that, The detection method satisfies one or more of the following conditions: The flow rate of the mobile phase is 0.9–1.1 ml / min; The column temperature of the chromatographic column is 25–35°C; The detection wavelength is 210-218 nm; The sample injection volume for the detection is 10–30 μl.

7. The detection method according to any one of claims 1-6, characterized in that, The detection method further includes the following solution preparation steps: Preparation of diluent: It is prepared by mixing water and acetonitrile, preferably by mixing water and acetonitrile in a volume ratio of 1:1; Preparation of test solution: Take 2,5-dichloro-4,6-dimethylnicotinonitrile and dilute quantitatively with diluent to prepare a solution containing approximately 0.2 mg per ml; Preparation of the reference solution: Take 2,5-dichloro-4,6-dimethylnicotinonitrile reference standard and dilute it quantitatively with diluent to prepare a solution containing approximately 0.4 μg per 1 ml; Preparation of system suitability solution: Take 2,5-dichloro-4,6-dimethylnicotinonitrile and each impurity reference standard, and dilute quantitatively with diluent to prepare a mixed solution containing approximately 0.2 mg of 2,5-dichloro-4,6-dimethylnicotinonitrile and 0.4 μg of each impurity per 1 ml.

8. The detection method according to any one of claims 1-7, characterized in that, The detection method further includes an analytical method validation step, comprising one or more of the following solution preparation steps: Preparation of APKP001 positioning solution: Take 2,5-dichloro-4,6-dimethylnicotinonitrile (APKP001) reference standard and dilute quantitatively with diluent to prepare a solution containing approximately 0.2 mg per 1 ml; Preparation of impurity stock solution: Take each impurity reference standard and dilute quantitatively with diluent to prepare a solution containing approximately 0.2 mg per 1 ml; Preparation of impurity localization solutions: Take each impurity stock solution separately and dilute it quantitatively with diluent to prepare a solution containing approximately 0.4 μg per 1 ml; Preparation of detection limit test solution: Take 1 ml each of APKP001 positioning solution and each impurity stock solution, place them in a 100 ml volumetric flask, dilute to the mark with diluent, shake well to obtain intermediate stock solution; dilute the intermediate stock solution stepwise with diluent until the peak height signal-to-noise ratio (S / N) of each component is approximately 3.

9. The detection method according to any one of claims 1-8, characterized in that, The content of relevant substances was calculated using the principal component self-comparison method with correction factors, and the calculation formula is as follows: Impurity content (%) = f × A 杂 / A 对 ×C 对 / C 供 ×100; In the formula: f is the correction factor for each impurity; A 杂 The peak area of ​​each impurity peak in the test solution; A 对 The peak area of ​​the main component peak in the control solution; C 对 The concentration of the control solution is (mg / ml). C 供 The concentration of the test solution is (mg / ml).

10. The application of the detection method according to any one of claims 1-9 in the quality control of opipcarbon or its starting materials.

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